Proteasome inhibitor PS-341 induced G2-M phase arrest and apoptosis in human glioblastoma multiforme

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The proteasome plays a pivotal role in the control of cell proliferation, apoptosis and differentiation in a variety of normal and tumor cells. PS-341, a novel boronic acid dipeptide that inhibits 20S proteasome activity, has been shown to have prominent activity both in vitro and in vivo systems, against various solid tumor types including breast, lung, colon, bladder, ovarian, pancreatic, prostate, and squamous cell cancers. Using 3 human glioblastoma multiforme (GBM) cell lines and 5 primary GBM explants, we examined the effect of PS-341 on their proliferation, cell cycle, apoptosis, as well as expression of proteins involved in cell growth. We also explored whether the effect of PS-341 on GBM cells is dependent on the mutational status of p53. PS-341 markedly inhibited growth of GBM cell lines and explants with a mean IC50 of PS-341 at about 1x10^-7 M. The cells developed a cell cycle block at G2/M with a concomitant decrease in S phase (≈ 2-fold), an increase of p21^WAF1, p27^KIP1 and a down-regulation of CDK2, CDK4 and E2F4. About 20 –40 % of the cells became apoptosis when exposed to 1x10^-7 M PS-341 for 24 hrs as shown by Tunel analysis. Immunoblot analysis of poly (ADP-ribose) polymerase (PARP) showed that a C-terminal 85 kDa apoptotic fragment was generated and levels of Bcl2 and Bcl-xl were decreased. These changes occurred in the cells irrespective of their p53 mutant status. One major effect of proteasome inhibition in many cell types is the inhibition of NF-kB translocation to the nucleus, due to blocking of the cytosolic degradation of Ik-B. We found that PS-341 decreased nuclear NF-kB and transcriptional activity of NF-kB as measured by a reporter gene containing NF-kB binding sites fused to the luciferase gene. We conclude that PS-341 has profound effects on GBM cells including inhibiting their cell growth and inducing their G2/M phase arrest, apoptosis and accumulation of p21^WAF1 and p27^KIP1 in a p53-independent fashion, as well as inhibiting NF-kB nuclear translocation and transcriptional activity. These findings suggested that PS-341 may be highly effective as a therapy for gliomas in patients.